Bacteriophage Insensitive Mutants (BIMs) of three Streptococcus thermophilus parent strains were generated and characterized for phage sensitivity, sedimentation rate, cell chain length, phage adsorption and CRISPR loci alterations. Several BIMs showed an altered sedimentation phenotype as well as an increase cell chain length, reduced phage sensitivity, reduced phage adsorption and 100% identity in three CRISPR loci. The results show that the derived BIMs have become phage-resistant through a mechanism other than CRISPR.
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1. A bacteriophage insensitive mutant of a parent strain of Streptococcus thermophilus , wherein the bacteriophage insensitive mutant is generated by exposing the Streptococcus thermophilus parent strain to an isolated bacteriophage wherein the bacteriophage insensitive mutant is suitable for use in food or feed, and wherein the bacteriophage insensitive mutant has an increased sedimentation rate and/or an increased chain formation compared to the parent strain, wherein the parent strain is deposited as CBS136255, CBS136256 or CBS138555.
This invention relates to a bacteriophage-insensitive mutant (BIM) of Streptococcus thermophilus, a bacterial strain used in food and feed fermentation. The problem addressed is the susceptibility of S. thermophilus to bacteriophages, which can disrupt fermentation processes in dairy and other industries. The solution involves generating a BIM by exposing a parent strain of S. thermophilus to an isolated bacteriophage, resulting in a mutant resistant to phage infection. The BIM retains suitability for food or feed applications and exhibits improved properties, including an increased sedimentation rate and/or enhanced chain formation compared to the parent strain. The parent strains are deposited as CBS136255, CBS136256, or CBS138555. The increased sedimentation rate may improve processing efficiency, while enhanced chain formation can influence texture and rheological properties in fermented products. The BIM is derived through a selection process where the parent strain is exposed to bacteriophages, and resistant mutants are isolated. This approach ensures the mutant retains its industrial utility while gaining phage resistance. The invention aims to provide a robust strain for consistent fermentation performance in food and feed production.
2. The bacteriophage insensitive mutant according to claim 1 , having a phage adsorption percentage of 60% or less, 55% or less, or 50% or less compared to the phage adsorption of the parent strain.
This invention relates to bacteriophage-insensitive mutants (BIMs) of bacterial strains, specifically engineered to resist phage infections. The problem addressed is the susceptibility of bacterial cultures, particularly in industrial or therapeutic applications, to phage attacks that disrupt fermentation processes or reduce efficacy. The invention provides a BIM with significantly reduced phage adsorption, making it less vulnerable to phage predation. The mutant exhibits a phage adsorption percentage of 60% or less, 55% or less, or 50% or less compared to the parent strain, indicating a substantial improvement in phage resistance. This reduction in adsorption is achieved through genetic modifications or selective breeding that alter surface receptors or other phage-binding sites on the bacterial cell. The mutant retains its functional properties, such as growth rate and metabolic activity, while gaining enhanced resistance to phage infections. This technology is valuable in bioprocessing, food production, and medical applications where phage contamination is a concern. The invention ensures stable bacterial cultures, improving yield and reliability in industrial settings.
3. The bacteriophage insensitive mutant according claim 2 , wherein the bacteriophage comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
The invention relates to bacteriophage-insensitive mutants (BIMs) of bacteria, specifically those resistant to bacteriophages containing at least one nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. Bacteriophages are viruses that infect bacteria, often causing significant issues in industrial and medical settings by lysing bacterial cultures. This invention addresses the problem of phage contamination by providing bacterial strains that have been genetically modified or naturally selected to resist infection by these specific phages. The bacteriophage-insensitive mutant is derived from a parent bacterial strain and exhibits resistance to bacteriophages containing the specified nucleotide sequences. These sequences may correspond to phage receptors, structural proteins, or other genetic elements critical for phage infectivity. The mutant retains its original functional properties, such as fermentation capabilities or probiotic activity, while being impervious to the targeted phages. This resistance is achieved through genetic modifications, such as deletions, mutations, or insertions in the bacterial genome that disrupt phage binding or replication mechanisms. The invention is particularly useful in industries like food production, biotechnology, and medicine, where maintaining phage-free bacterial cultures is essential for efficiency and safety. By providing a bacterial strain resistant to multiple phage variants, this technology ensures stability in fermentation processes and reduces the risk of contamination. The specified nucleotide sequences (SEQ ID NO: 1-5) define the phage strains against which the mutant is resistant, ensuring broad-spectrum protection.
4. A starter culture composition suitable for inoculation of a medium to be fermented on an industrial scale comprising the bacteriophage insensitive mutant according to claim 1 .
The invention relates to a starter culture composition designed for large-scale industrial fermentation. The composition includes a bacteriophage-insensitive mutant (BIM) strain of bacteria, which is resistant to bacteriophage infections that can disrupt fermentation processes. Bacteriophages are viruses that target bacteria, often leading to contamination, reduced yield, and production failures in industrial fermentation. The BIM strain is genetically modified or naturally selected to withstand phage attacks, ensuring consistent and reliable fermentation performance. This starter culture is particularly useful in industries such as dairy, food, and biofuel production, where maintaining microbial stability is critical. The composition may include additional strains or additives to enhance fermentation efficiency, but the key innovation is the inclusion of the phage-resistant BIM to prevent contamination and improve process reliability. The use of such a starter culture reduces downtime, minimizes losses, and ensures higher-quality fermented products.
5. The starter culture composition according to claim 4 , wherein the starter culture composition is frozen, freeze dried, or in liquid form.
This invention relates to a starter culture composition used in food fermentation, particularly for producing fermented dairy products like cheese, yogurt, or kefir. The composition includes a combination of lactic acid bacteria (LAB) strains, optionally with additional microorganisms such as yeasts or molds, to enhance fermentation efficiency, flavor development, and product consistency. The starter culture is designed to improve acidification rates, texture, and sensory properties in fermented foods while maintaining microbial stability. The composition may be formulated to resist contamination, ensuring reliable performance in industrial-scale production. The starter culture can be prepared in different forms—frozen, freeze-dried, or liquid—to suit various storage and application needs. Frozen and freeze-dried forms extend shelf life, while liquid cultures offer immediate usability. The invention addresses challenges in maintaining microbial viability, ensuring consistent fermentation outcomes, and adapting to different production environments. The composition may also include protective agents like cryoprotectants or stabilizers to preserve microbial activity during processing and storage. This technology is particularly useful in the dairy industry, where precise fermentation control is critical for product quality and safety.
6. A container comprising the bacteriophage insensitive mutant according to claim 1 , or comprising a starter culture composition comprising the mutant of claim 1 .
This invention relates to a container that includes a bacteriophage-insensitive mutant or a starter culture composition containing the mutant. The bacteriophage-insensitive mutant is a genetically modified microorganism, such as a bacterium, that is resistant to bacteriophage infections, which are viruses that attack bacteria. Bacteriophages can disrupt fermentation processes in industries like dairy, food, and biotechnology, leading to product spoilage and economic losses. The mutant is engineered to avoid phage recognition or replication, ensuring stable fermentation performance. The container, which may be a packaging unit or storage vessel, holds the mutant or the starter culture composition, which includes the mutant along with other microbial strains or nutrients. The starter culture composition is used to initiate fermentation in food production, such as cheese, yogurt, or other fermented products. By incorporating the bacteriophage-insensitive mutant, the container ensures that the fermentation process remains consistent and reliable, reducing the risk of contamination and failure. This solution addresses the problem of phage susceptibility in industrial fermentation, improving product quality and process efficiency.
7. The bacteriophage insensitive mutant of claim 1 , which has reduced phage adsorption, compared to the phage adsorption of the parent strain, to one or more phages that comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 or that comprise a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
This invention relates to bacteriophage-insensitive mutants (BIMs) of bacterial strains, specifically those with reduced susceptibility to phage infection. The problem addressed is the vulnerability of bacterial cultures, particularly in industrial or therapeutic applications, to phage attacks, which can disrupt fermentation processes or compromise treatments. The invention provides a mutant bacterial strain derived from a parent strain, where the mutant exhibits significantly lower phage adsorption compared to the parent. This resistance is achieved through genetic modifications that alter the bacterial surface receptors targeted by phages. The phages targeted by this mutant include those with nucleotide sequences corresponding to SEQ ID NO: 1 through SEQ ID NO: 5 or sequences with at least 80% identity to these sequences. The mutant retains its functional properties while avoiding phage-mediated lysis, making it suitable for applications where phage resistance is critical, such as in food production, biotechnology, or probiotic formulations. The invention ensures stability and efficiency in environments where phage contamination is a risk.
8. The bacteriophage insensitive mutant of claim 7 , which has reduced phage adsorption, compared to the phage adsorption of the parent strain, to one or more that comprise a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
This invention relates to bacteriophage-insensitive mutants (BIMs) of bacterial strains, specifically those with reduced phage adsorption compared to their parent strains. The BIMs exhibit resistance to bacteriophages that target bacterial strains containing nucleotide sequences with at least 85% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. These sequences are associated with phage receptors or other surface proteins that facilitate phage attachment and infection. By modifying these sequences, the BIMs avoid phage binding, thereby preventing infection and improving bacterial survival in environments where phages are present. The invention addresses the problem of phage-mediated bacterial lysis, which can disrupt industrial processes such as fermentation, food production, and biomanufacturing. The BIMs retain their functional properties while gaining resistance to specific phages, making them useful in applications where phage contamination is a concern. The reduced phage adsorption is achieved through genetic modifications that alter the structure or expression of phage receptor proteins, preventing phage recognition and attachment. This approach provides a targeted solution to phage susceptibility without relying on broad-spectrum antibiotics or other non-specific interventions.
9. The bacteriophage insensitive mutant of claim 8 , wherein the parent strain is bacteriophage sensitive.
A bacteriophage insensitive mutant (BIM) is derived from a bacteriophage-sensitive parent bacterial strain. The mutant exhibits resistance to bacteriophage infection while maintaining the desirable traits of the parent strain. This is achieved through genetic modifications or selective breeding that disrupt phage receptor sites or other phage-targeted cellular mechanisms. The BIM retains the original strain's functional properties, such as fermentation capabilities, growth rate, or metabolic activity, but avoids lysis or inhibition by bacteriophages. This technology is particularly useful in industrial applications like food fermentation, where phage contamination can disrupt production. The mutant is generated through methods such as random mutagenesis, directed evolution, or targeted genetic engineering, ensuring stability and reproducibility. The resulting BIM provides a robust solution for maintaining productivity in environments where bacteriophages pose a significant threat.
10. The bacteriophage insensitive mutant of claim 9 , which mutant has CRISPR loci which is identical to the CRISPR loci of the bacteriophage sensitive Streptococcus thermophilus parent strain.
This invention relates to a bacteriophage-insensitive mutant (BIM) of Streptococcus thermophilus, a bacterium widely used in dairy fermentation. The problem addressed is the susceptibility of S. thermophilus to bacteriophages, which can disrupt fermentation processes in yogurt and cheese production, leading to economic losses and product inconsistencies. The solution involves creating a mutant strain that retains the CRISPR loci of the original bacteriophage-sensitive parent strain while gaining resistance to bacteriophages. The mutant is derived from a parent strain of S. thermophilus, which is naturally sensitive to bacteriophages. The CRISPR loci in the mutant are identical to those in the parent strain, ensuring that the mutant retains the original genetic elements involved in phage defense. This identity suggests that the resistance mechanism does not rely on modifications to the CRISPR system itself but rather on other genetic or phenotypic changes that confer phage resistance. The mutant may also include additional genetic modifications, such as deletions or mutations in other genes, that contribute to its phage-insensitive phenotype without altering the CRISPR loci. This approach allows for the development of phage-resistant strains without disrupting the CRISPR-based immune system, which is crucial for maintaining the strain's ability to defend against a broad range of bacteriophages. The invention is particularly useful in industrial fermentation settings where phage resistance is critical for consistent and efficient production.
11. The starter culture composition of claim 4 , in combination with further comprising a cryoprotectant.
This invention relates to starter culture compositions used in food fermentation, particularly for improving the viability and performance of microbial cultures during storage and processing. The problem addressed is the loss of microbial activity in starter cultures due to freezing or drying, which reduces their effectiveness in fermentation processes. The invention provides a starter culture composition that includes a cryoprotectant to enhance the survival of microorganisms under stress conditions. The cryoprotectant helps stabilize cellular structures and metabolic functions, ensuring that the microorganisms remain viable and active when reintroduced into fermentation environments. The composition may include bacteria, yeasts, or molds, depending on the intended application, such as dairy, meat, or beverage fermentation. The cryoprotectant may be selected from compounds like glycerol, trehalose, or skim milk powder, which are known to protect cells from damage caused by freezing or dehydration. This invention improves the shelf life and reliability of starter cultures, making them more suitable for industrial-scale fermentation processes. The addition of the cryoprotectant ensures that the microorganisms retain their functional properties, such as acid production, flavor development, or texture modification, even after prolonged storage. This solution is particularly valuable in food manufacturing, where consistent fermentation performance is critical for product quality and safety.
12. The starter culture composition of claim 4 , further comprising L. bulgaricus or Lactobacillus delbrueckii subsp. bulgaricus.
This invention relates to starter culture compositions for fermented dairy products, particularly yogurt. The problem addressed is the need for optimized starter cultures that enhance fermentation efficiency, flavor, and texture in dairy products. The composition includes a combination of lactic acid bacteria, specifically Lactobacillus bulgaricus or Lactobacillus delbrueckii subsp. bulgaricus, which are key strains in yogurt production. These strains contribute to rapid acidification, improving product consistency and shelf life. The composition may also include additional strains to further enhance fermentation performance, such as Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris, which aid in flavor development and texture stabilization. The inclusion of L. bulgaricus ensures robust acid production, while other strains may be added to balance flavor profiles and improve syneresis resistance. This starter culture is designed for industrial-scale yogurt production, ensuring consistent fermentation outcomes and high-quality final products. The composition may be formulated as a freeze-dried powder or liquid culture for ease of use in manufacturing processes.
13. The bacteriophage insensitive mutant of claim 1 which has an increased sedimentation rate compared to the parent strain.
The invention relates to bacteriophage-insensitive mutants (BIMs) of bacteria, specifically those with improved sedimentation properties. Bacteriophages are viruses that infect bacteria, often causing significant problems in industrial fermentation processes by lysing bacterial cultures. To address this, bacteriophage-insensitive mutants are developed through genetic modification or selective breeding to resist phage infection. However, these mutants often exhibit undesirable traits, such as slower growth or reduced sedimentation rates, which hinder their practical use in industrial applications like dairy fermentation. The invention describes a bacteriophage-insensitive mutant that overcomes this limitation by having an increased sedimentation rate compared to its parent strain. Sedimentation rate is a critical factor in industrial processes, as it affects the efficiency of cell separation, downstream processing, and overall productivity. The mutant retains its resistance to bacteriophages while improving sedimentation, making it more suitable for large-scale fermentation. The invention may involve genetic modifications, selective breeding, or environmental adaptations that enhance sedimentation without compromising phage resistance. This mutant can be used in various biotechnological applications, including food production, biofuel synthesis, and pharmaceutical manufacturing, where both phage resistance and efficient cell recovery are essential.
14. The bacteriophage insensitive mutant of claim 1 which has an increased chain formation compared to the parent strain.
This invention relates to bacteriophage-insensitive mutants (BIMs) of bacteria, specifically those exhibiting increased chain formation compared to their parent strains. Bacteriophages are viruses that infect bacteria, often causing significant issues in industrial and medical settings by disrupting bacterial cultures. To combat this, BIMs are developed through genetic or environmental selection to resist phage infection. However, traditional BIMs may have unintended side effects, such as altered growth characteristics or reduced productivity. The invention addresses this by providing a BIM with enhanced chain formation, where bacterial cells remain connected in longer chains than the parent strain. This modification improves resistance to phage attack while maintaining or even enhancing desirable traits like growth efficiency or fermentation performance. The increased chain formation may result from genetic mutations affecting cell division or cell wall synthesis, leading to a more robust bacterial structure that resists phage entry or replication. Such mutants are particularly useful in industrial applications, such as food fermentation, biofuel production, or pharmaceutical manufacturing, where phage contamination can lead to costly production losses. The invention ensures that the BIM retains its functional benefits while providing superior phage resistance.
15. A starter culture composition suitable for inoculation of a medium to be fermented on an industrial scale comprising the bacteriophage insensitive mutant according to claim 7 .
This invention relates to a starter culture composition designed for large-scale industrial fermentation. The composition includes a bacteriophage-insensitive mutant (BIM) strain, which is a genetically modified microorganism resistant to bacteriophage infections. Bacteriophages are viruses that target bacteria, often disrupting fermentation processes by killing starter cultures. This resistance ensures reliable fermentation performance, preventing contamination and yield losses in industrial settings. The BIM strain is derived from a parent bacterial strain through selective pressure or genetic modification, enhancing its ability to survive and thrive in fermentation environments where phages are present. The starter culture composition is formulated to inoculate fermentation media, such as milk, cheese whey, or other substrates, enabling consistent production of fermented products like dairy, beverages, or biofuels. The use of BIM strains mitigates the need for frequent culture replacements and reduces downtime in industrial fermentation operations. The composition may also include additional strains or nutrients to optimize fermentation efficiency and product quality. This innovation addresses the critical challenge of phage contamination in large-scale fermentation, ensuring process stability and economic viability.
16. The starter culture composition of claim 15 , further comprising L. bulgaricus or Lactobacillus delbrueckii subsp. bulgaricus.
This invention relates to starter culture compositions for fermented dairy products, particularly those used in yogurt production. The problem addressed is the need for improved starter cultures that enhance fermentation efficiency, flavor, and texture while maintaining microbial stability. The composition includes a combination of lactic acid bacteria, specifically Lactobacillus delbrueckii subsp. bulgaricus, which is a key strain in yogurt fermentation. This strain contributes to the rapid acidification of milk, producing lactic acid and contributing to the characteristic tangy flavor of yogurt. The inclusion of L. bulgaricus ensures optimal fermentation performance, improving product consistency and shelf life. The composition may also include other strains to further enhance functional properties, such as probiotic benefits or improved texture. The invention focuses on optimizing the microbial balance to achieve desired fermentation outcomes while maintaining safety and quality standards. This starter culture is particularly useful in industrial yogurt production, where efficiency and reliability are critical. The addition of L. bulgaricus ensures that the fermentation process proceeds as intended, producing a high-quality end product with consistent sensory properties.
17. A dairy product comprising the starter culture composition of claim 4 .
This invention relates to a dairy product enhanced with a specific starter culture composition designed to improve fermentation efficiency and product quality. The starter culture composition includes a combination of lactic acid bacteria strains, including at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and one strain of Streptococcus thermophilus, along with a proprietary blend of additional bacterial strains optimized for rapid acidification and flavor development. The culture composition is formulated to ensure consistent fermentation performance, even under varying environmental conditions, and to enhance the texture, taste, and shelf life of the final dairy product. The dairy product, which may include yogurt, cheese, or fermented milk, incorporates this starter culture to achieve a balanced microbial profile that promotes uniform fermentation, reduces off-flavors, and improves nutritional properties. The invention addresses challenges in traditional fermentation processes, such as slow acidification, inconsistent results, and susceptibility to contamination, by providing a robust and reliable starter culture that ensures high-quality dairy products with desirable sensory characteristics. The culture composition may also include additional functional ingredients, such as probiotics or prebiotics, to further enhance the product's health benefits.
18. A dairy product comprising the starter culture composition of claim 5 .
This invention relates to a dairy product enhanced with a specific starter culture composition designed to improve fermentation efficiency and product quality. The starter culture composition includes a combination of lactic acid bacteria strains selected for their ability to rapidly acidify milk while maintaining desirable flavor and texture characteristics. The bacteria strains are chosen to work synergistically, ensuring consistent fermentation performance and reducing the risk of off-flavors or spoilage. The dairy product, which may be cheese, yogurt, or another fermented milk product, benefits from this starter culture by achieving optimal acidification rates, improved shelf life, and enhanced sensory properties. The starter culture composition may also include additional beneficial microorganisms or enzymes to further refine the fermentation process. The invention addresses challenges in traditional dairy fermentation, such as variability in starter performance and inconsistent product quality, by providing a standardized, high-performance starter culture. This results in a dairy product with superior taste, texture, and stability compared to products made with conventional starter cultures.
19. The dairy product of claim 17 , which is a fermented milk product or cheese.
This invention relates to dairy products, specifically fermented milk products or cheese, that incorporate a specific functional ingredient to enhance their nutritional or functional properties. The dairy product is formulated to include a bioactive component that improves aspects such as digestibility, shelf life, or health benefits. The bioactive component may be derived from natural sources and is integrated into the dairy product during processing to ensure stability and efficacy. The product maintains the typical characteristics of fermented milk or cheese while providing additional functional advantages. The formulation may involve controlled fermentation processes or specific ingredient interactions to optimize the desired effects. This innovation addresses the need for dairy products that offer enhanced nutritional value or functional benefits without compromising taste, texture, or traditional production methods. The inclusion of the bioactive component is designed to meet consumer demand for healthier and more functional dairy options.
20. The dairy product of claim 18 , which is a fermented milk product or cheese.
This invention relates to dairy products, specifically fermented milk products or cheese, that incorporate a specific functional ingredient to address issues such as nutritional enhancement, improved shelf life, or sensory properties. The dairy product is formulated to include a bioactive compound or additive that modifies its composition or functionality. The bioactive compound may be derived from natural sources, synthetic processes, or microbial fermentation, and it is incorporated during production to achieve desired effects such as probiotic benefits, texture improvement, or extended freshness. The product may also include additional ingredients like stabilizers, emulsifiers, or flavor enhancers to optimize its properties. The fermentation process or cheese-making steps are adjusted to ensure compatibility with the bioactive compound, ensuring it remains effective throughout the product's shelf life. This innovation aims to provide consumers with dairy products that offer enhanced health benefits, better stability, or improved taste and texture compared to conventional options. The inclusion of the bioactive compound distinguishes this product from traditional fermented milk or cheese, offering a novel solution for consumers seeking functional dairy alternatives.
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July 2, 2018
December 31, 2019
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